Biolever mini tips

Samples were immobilized to AFM discs with epoxy. Air tapping mode measurements were taken with Tap300Al-G tips (BudgetSensors), with a nominal spring constant 40 N m⁻¹ at a scan rate of 2 Hz. Liquid AFM was performed on the Asylum Cypher in Fast Force Mapping mode with BioLever Mini tips (Olympus Corporation), with a nominal spring constant of 0.09 N m⁻¹ at a set point of 250 pN and a Z rate of 299.40 Hz.
Wrinkle radii of curvature were determined by fitting a quadratic function to multiple line profiles of peaks and valleys and are reported as absolute values. Protein surface coverage was quantified by analysis of AFM images in Gwyddion software. A 140 × ∼800 nm region along each peak or valley was cropped, and a second-order flattening was performed to remove the curvature of the substrate's topography. A threshold with user selected cut-off was used to separate protein from substrate and quantify the surface coverage of the protein. Standard error was calculated by averaging measurements from 3 regions on the same sample. A t test was performed to determine significance of differences in peak/valley coverage, with p ≤ 0.05 being considered significant. Fast Fourier Transform (FFT) filtering was performed in Gwyddion software.

DNA molecules were immobilized for liquid imaging using Ni²⁺ as previously reported (36 (link),37 (link)). A 20 μl solution containing 0.3 nM of DNA in 100 mM NaCl, 10 mM Tris–HCl pH 8 and 7.5 mM MgCl₂ was first deposited on freshly-cleaved mica (grade V-4 mica from SPI) and incubated for 60 s to allow molecules to equilibrate. Then, 2 μl of 2.5 mM NiCl₂ were added to the solution and incubated for additional 60 s to fix the molecules to the mica surface. The sample was then rinsed four times with 80 μl imaging buffer 10 mM Tris–HCl pH 8 and 10 mM KCl. Images were obtained in a final volume of 80 μl. Images were taken with an AFM from Nanotec Electronica S.L. with Biolever mini tips (AC-40TS Olympus, tip stiffness 0.02–0.1 N/m, tip radius ∼10 nm) using amplitude modulation imaging mode with a free amplitude of 1–2 nm and a set point of ∼0.8 nm.
Images obtained in air and in liquid were taken at a resolution of 1.46 nm/pixel and processed using the ‘Flatten plus’ utility of the WSxM freeware (38 (link),39 (link)). Contour lengths were computed by manually tracing the molecules using WSxM. Persistence length of molecules imaged in air were calculated using the tracing routine described in (34 (link),40 (link)) by taking 290 nm traces with 2.5 nm point-to-point (l) separation. Persistence length of molecules imaged in liquid were obtained by taking 292 nm traces with l = 4.0 nm.